The steady state level of a messenger RNA (mRNA) is a function of its rate of synthesis and degradation. Just as numerous differences in the rate of synthesis (i.e., transcription) exist, so to do differences in relative mRNA stability. Messenger RNAs with very short half-lives include many clinically important transcripts, namely transcription factors, growth factors and cytokines. Messenger RNAs with extremely long half-lives also have been described, including globin transcripts. In addition, messenger RNAs from some disease alleles contain an inappropriately placed nonsense codon resulting in premature termination of the open frame encoding the protein product. These “nonsense codon-containing” mRNAs are degraded very rapidly in cells. Clearly, mRNA stability plays an important regulatory role in the molecular biology of the cell.
Since mRNA stability plays a key role in regulated gene expression, it is a natural target for drug development. This potential for drug development is further enhanced when one surveys some of the best described cases of regulated mRNA stability. First, the importance of modulation of the immune system is central to many aspects of clinical medicine. Several genes that play a key role in immunologic responses and development (e.g. cytokines) are clearly regulated at the level of mRNA stability. Second, the expression of several proteins that play regulatory roles in the cell cycle are also regulated at the level of mRNA stability. This observation implies that mRNA stability may influence cancer treatments, for example regulation of mRNA turnover can control the expression of a variety of growth factors and proto-oncogenes. Finally, all aspects of gene therapy would benefit from a better understanding of modulating influences of mRNA stability. Messenger RNA stability, therefore, remains an unexploited target for drug development with a great deal of potential.
Messenger RNAs contain a poly(A) tail of approximately 200 bases at their 3′ end when they are synthesized. The first step in the pathway of turnover of mRNAs in mammalian cells appears to be the stepwise removal of the tail by a process called deadenylation. The rate of deadenylation, as well as the overall rate of turnover of the mRNA, is influenced by sequence elements present in the mRNA. The best characterized of these elements to date is an “AU rich” element located in the 3′ untranslated region of the mRNA body. The presence of an AU rich element in a mRNA causes a dramatic increase in the rate of deadenylation/degradation, effectively shortening the half-life of a mRNA. After the poly(A) tail is shortened to a minimal length, the body of the transcript is rapidly degraded with no apparent intermediates. Certain aspects of the foregoing are described in co-pending application Ser. No. 09/320,609, filed May 26, 1999, incorporated herein by reference in its entirety.
In the yeast Saccharomyces cerevisiae, deadenylated mRNAs are usually degraded by two major pathways. In the predominant pathway, deadenylated mRNAs are decapped and degraded by a 5′-to-3′ exonuclease. If this pathway is blocked by genetic mutations, an alternative pathway can be observed in which deadenylated mRNAs are degraded by a 3′-to-5′ exonuclease.
It is not known how mammalian mRNAs are degraded in cells following their deadenylation nor how the process is regulated. Only one study has been performed to date that suggests the possibility that mammalian mRNAs can be decapped in vivo. This study, however, used an indirect highly-sensitive PCR assay that may not have detected true intermediates in the mRNA degradation pathway. There is currently no direct biochemical evidence for decapping in mammalian cells. The putative human homologue of the yeast decapping enzyme Dcp1p surprisingly does not possess detectable decapping activity and has recently been identified as a transcription factor.
The lack of mechanistic detail in the model for mRNA turnover presented above reflects the lack of a good experimental approach to study the process. Human cells and mammals in general do not represent a genetic system that is easily exploited using current technology. The key to understanding mechanisms of gene expression in cells from higher organisms, therefore, lies in a biochemical approach. When used in conjunction with reagents developed from chemical or molecular approaches, such systems can provide the backbone of assays to understand and exploit aspects of cellular biology for therapeutic advantages.
Decapping is a major regulated step in the turnover of yeast mRNAs. It is a key regulatory element in mammalian cells as well. As shown herein, AU-rich instability elements stimulate decapping efficiency. Several other elements have been identified in mammalian mRNAs that stabilize transcripts. A 51 base pyrimidine-rich element has been identified in the relatively stable alpha-globin mRNA that is responsible for its extremely long half-life in vivo. As shown herein, such stability elements also regulate decapping efficiency. Regulation of mRNA decapping, therefore, plays an important role in the post transcriptional regulation of gene expression.
It is toward the development of an in vitro system in which mammalian messenger RNA decapping occurs and can be exploited for the screening of decapping modulators, for identifying species-specific decapping enzymes and associated factors, among other uses, that the present invention is directed.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.